The present invention relates to a cDNA encoding a plant-derived hydrolase, a gene encoding the same, a plasmid vector carrying said cDNA and a transformant.
Epoxide hydrolase is an enzyme involved in the biological protective system functioning for preventing chemical reactions hazardous for living organisms in such a manner that the enzyme hydrolyses hazardous epoxide compounds generated through peroxidation in living organisms, to eliminate the high chemical reactivity of the epoxide compounds.
It is evidenced that epoxide hydrolase is present in animal cells and plant cells. Particularly, epoxide hydrolase derived from plants is expected to be applicable to agricultural fields and food industries. Because the content thereof is very low, the mass-scale production thereof has been believed to be extremely difficult.
As to genetic information concerning epoxide hydrolase, currently, reports have been issued about the genetic information thereof from animal cells, such as human liver cell [Beetham, J. K. et al., Arch. Biochem. Biophys., 305, 197-201 (1993)] and liver cells from rats and mice [Knehr, M. et al., J. Biol. Chem., 268, 17623-17627 (1993); Grant, D. F. et al., J. Biol. Chem., 268, 17628-17633 (1993)].
Meanwhile, the genetic information thereof from plant cells including potato [Stapleton, A. et al., Plant J. 6, 251-258 (1994)] and Arabidopsis [Kiyosue, T. et al., Plant J., 6, 259-269 (1994)] has been reported as well.
However, no report regarding genetic information about epoxide hydrolase derived from plants has been published yet, except the aforementioned reports. No plant-derived epoxide hydrolase with high activity has been reported yet.
The inventors have made attempts to purify a plant-derived epoxide hydrolase and to clone the cDNA and gene of the epoxide hydrolase based on the amino acid sequence thereof. Consequently, the inventors have successfully identified the whole structure of the cDNA of the enzyme and that of the gene thereof and have also achieved successfully the construction of an expression system in Escherichia coli by utilizing the cDNA and the gene. Thus, the invention has been achieved.
Based on the analysis of the amino acid sequence of the purified epoxide hydrolase, synthetic degenerate oligonucleotides were prepared. By subsequently extracting MRNA from a fully ripened soy seed and synthesizing a double-stranded cDNA using the mRNA, which was then integrated via an adapter into a phage vector, a cDNA library was constructed.
By labeling the prepared synthetic oligonucleotides and using the labeled synthetic oligonucleotides as probes, cDNA encoding the epoxide hydrolase of its full length was cloned by screening from the aforementioned cDNA library. The analysis of the primary structure of the cDNA indicated the whole amino acid sequence of the epoxide hydrolase including the signal peptide.
The epoxide hydrolase gene was cloned by using a commercially available gene library prepared by partially digesting nuclear DNA extracted and purified from sprout-developed soy with a restriction endonuclease Mbo I and integrating the digestion product in a phage vector.
By labeling the preliminarily recovered cDNA of the epoxide hydrolase and using the labeled cDNA as probe for screening from the aforementioned gene library, the epoxide hydrolase gene including the 5xe2x80x2 upstream region was cloned.
Using the cloned epoxide hydrolase cDNA to construct an Escherichia coli expression system by utilizing a promoter T7, the present enzyme was successfully expressed while a simple and rapid purification method thereof was also established.
More specifically, a first aspect of the invention relates to a cDNA encoding the plant-derived epoxide hydrolase having the amino acid sequence of SQ ID No. 1 in the Sequence Listing.
A second aspect of the invention relates to a gene encoding the plant-derived epoxide hydrolase having the amino acid sequence of SQ ID No. 2 in the Sequence Listing.
A third aspect of the invention relates to a plasmid vector carrying the cDNA encoding the plant-derived epoxide hydrolase in the first aspect of the invention.
A fourth aspect of the invention relates to a transformant (FERM BP-6624) retaining the plasmid vector in the third aspect of the invention.
The invention will now be described in more detail hereinbelow.
As described in the column of xe2x80x9cDescription of the Related Artxe2x80x9d, the epoxide hydrolase of the invention is an enzyme with an action hydrolyzing epoxide hazardous to living organisms to generate diol and the like.
The first aspect of the invention is now described.
The first aspect of the invention relates to the cDNA encoding the plant-derived epoxide hydrolase having the amino acid sequence of SEQ ID No. 1 in the Sequence Listing.
The inventors have succeeded to obtain the cDNA of the first aspect of the invention in the following manner.
1. Preparation of cDNA Library
1.1 Extraction and Purification of Epoxide Hydrolase in Plants
Epoxide hydrolase is present in cells of various plants and animals. In the first aspect of the invention, epoxide hydrolase derived from the cells of a plant is specifically used.
The cells of a plant are preferably cells of any seed with abundance of epoxide hydrolase, but are not limited thereto.
For extracting epoxide hydrolase from soy seed, use is preferably made of soy between early growing stage and ripe stage, preferably soy between early growing stage and later growing stage.
So as to obtain epoxide hydrolase, a raw material plant should necessarily be purified at a high level. The raw material plant includes for example soy, Arabidopsis, and potato.
The method for purifying the enzyme is illustrated in a case using soy as the raw material. Grinding the plant seed between early growing stage and ripe stage, and adding an appropriate buffer solution to the ground seed to extract the soluble fraction, the fraction is subjected to dialysis to separate the liquid fraction from the solid fraction. The resulting crude enzyme solution is applied to hydrophobic chromatography or gel filtration, whereby almost pure epoxide hydrolase can be recovered.
1.2 Analysis of Inner Amino Acid Sequence of Purified Epoxide Hydrolase
The purified epoxide hydrolase obtained in 1.1 cannot be analyzed as such of its inner amino acid sequence because the N terminus thereof is blocked. Therefore, the following procedures should be carried out.
Enzymatically degrading the epoxide hydrolase into short peptides and fractionating samples of the individual peptides by high performance liquid chromatography, the amino acid sequence of each of the samples is determined. The amino acid sequence is conveniently determined by Edman degradation using an automatic amino acid sequencer.
The individual samples are examined of their amino acid sequences; oligonucleotides are synthesized by using selected regions with less degeneracy among them; and the resulting oligonucleotides are used as the following probes.
1.3 Preparation of Poly(A)+ RNA
RNA is then extracted from the plant seed, to prepare poly(A)+ RNA from the extracted total RNA.
The poly (A)+ RNA can be prepared from the plant by using the SDS-phenol method according to the method by Fukazawa, C. et al., Journal of Biological Chemistry, 200, 6234-6239 (1985).
1.4 Construction of cDNA Library
Synthetically preparing a double-stranded cDNA by using the resulting mRNA and integrating the cDNA via an adapter in a phage vector, a cDNA library is constructed.
The cDNA library can be prepared satisfactorily by various methods including for example the Okayama-Berg method [Okayama, H. and Berg, P., Mol. Cell Biol., 2, p. 161 (1982)] and the Gubler-Hoffman method [Gubler, U. and Hoffman, B. J., Gene, 25, p.263 (1983)]; and for simplicity, the latter method is preferable.
The Gubler-Hoffman method is now described below.
First, a double-stranded cDNA is synthetically prepared by using the poly(A)+ RNA. To the cDNA is ligated an adapter carrying digestion sites with restriction endonucleases such as Eco RI, Not I and BamH I. By polyacrylamide gel electrophoresis, cDNAs of 500 bp or more are scissored out and collected from the gel, while excessive such adapter is removed.
Subsequently, a phosphate group is inserted at the 5xe2x80x2 termini of the resulting nucleotide sequences, which are then restriction digested and ligated to the dephosphorylated xcexgt10 phage vector arm (manufactured by TaKaRa Brewery, Co.). The resulting nucleotide sequences are packaged in a xcex phage. In such manner, a cDNA library is prepared.
2. Cloning of Plant-derived Epoxide Hydrolase cDNA of Full Length from cDNA Library and Determination of Nucleotide sequence thereof
2.1 Cloning of Full-length Epoxide Hydrolase cDNA
Labeling the oligonucleotides obtained above in 1.2 and using the resulting oligonucleotides as probes, the cloning of the plant-derived epoxide hydrolase cDNA of full length is accomplished by screening from the cDNA library described in 1.4.
The screening from the cDNA library is successfully executed by separating plaques positive at hybridization with plaques of about 1,000,000 in number, using, as probes, the radio-labeled synthetic oligonucleotides [Fukazawa, C. et al., Journal of Biological Chemistry, 200, 6234-6239 (1985)].
Purifying the resulting positive plaques and allowing Escherichia coli to be infected with the plaques to proliferate the phage, the phage particle is purified to obtain phage DNA. The phage DNA can be purified by ultra-filtration method on a CsCl step-wise density gradient.
2.2 Structure Determination of Recovered Phage DNA
From the purified phage DNA is cleaved the insert with a restriction enzyme, which is then purified. The insert is sub-cloned in a plasmid vector, for DNA sequencing. Consequently, the cDNA encoding the amino acid sequence of SEQ ID No. 1 in the Sequence Listing was obtained. The analysis of the cDNA suggested that the cDNA of the positive plaque cloned encoded the full-length epoxide hydrolase. More specifically, the cDNA is according to the first aspect of the invention.
The cDNA composed of 1,332 bp in its full length encodes 341 amino acids in total from the starting methionine.
The second aspect of the invention is now described.
The second aspect of the invention relates to the gene encoding the plant-derived epoxide hydrolase having the amino acid sequence of SEQ ID No:4 in the Sequence Listing.
The gene of the second aspect of the invention can be obtained on the basis of the cDNA of the first aspect of the invention. Continuously subsequent to the identification of the cDNA sequence in the first aspect of the invention, the inventors have selected the following steps.
3. Cloning of Plant-derived Epoxide Hydrolase Gene of Full Length and Determination of Nucleotide Sequence Thereof
Through screening from a commercially available soy gene library (Stratagene Ltd.) using the labeled cDNA of the first aspect of the invention as probe, the cloning of the plant-derived epoxide hydrolase gene of its full length is accomplished. The cloning is successfully achieved through the aforementioned plaque hybridization, to subsequently select positive phages.
The soy gene library purchased from the commercial source has been prepared by extracting and purifying nuclear DNA from sprout-developed soy and partially digesting the nuclear DNA with a restriction endonuclease Mbo I, and thereafter integrating the digestion product in a phage vector.
Purifying the resulting positive phages and allowing the phages to be subcloned in a plasmid vector, the DNA was sequenced by the same method as described above in 2.2. Consequently, the nucleotide sequence of the plant-derived epoxide hydrolase gene and the amino acid sequence encoded thereby can be recovered in accordance with the second aspect of the invention (see SEQ ID No: 2 in the Sequence Listing).
The sequence is composed of 1,933 bp in its full length (see SEQ ID No: 3 in the Sequence Listing), wherein two introns divide the sequence into three exons.
The individual introns, namely first intron and second intron, are composed of 168 bp and 148 bp, respectively, in nucleotide number. (See SEQ ID No: 3 in the Sequence Listing.)
The third aspect and fourth aspect of the invention will now be described collectively.
The third aspect of the invention relates to a plasmid vector carrying the cDNA encoding the plant-derived epoxide hydrolase in the first aspect of the invention.
The fourth aspect of the invention relates to a transformant retaining the plasmid vector in the third aspect of the invention.
In other words, the third and fourth aspects of the invention provide an expression system of the plant-derived epoxide hydrolase.
The plasmid vector and transformant described in the third and fourth aspects, respectively, of the invention are prepared by the following procedures.
4. Preparation of Plasmid Vector and Transformant
4.1 PCR Amplification of cDNA Nucleotide Sequence in the First Aspect of the Invention
Based on the cDNA nucleotide sequence in the first aspect of the invention (see SEQ ID No. 1 in the Sequence Listing) and the ion-spray mass spectrometry of the purified epoxide hydrolase, a 32-mer oligonucleotide primer containing a restriction Nde I site at the 5xe2x80x2 terminus (N terminus) and a 34-mer oligonucleotide primer containing a restriction Eco RI site at the 5xe2x80x2 terminus (C terminus), are synthesized (see SEQ ID Nos: 6 and 7 in the Sequence Listing). Restriction enzymes suitable for the expression vectors described below can satisfactorily be used, so the enzymes are not limited to the restriction enzymes described above.
PCR was conducted by using these primers and using the plant-derived epoxide hydrolase cDNA of the first aspect of the invention as template. PCR can be performed, for example, under the conditions described in Example 3, with no specific limitation.
4.2 Preparation of Plasmid Vector and Transformant
The band recovered in 4.1 was sub-cloned in a TA vector [pCR2.1] (manufactured by Invitrogen Co.) and was then subjected to DNA sequencing by the same method as described in 2.2, whereby it was confirmed that no mutation occurred in the expression cDNA amplified by PCR.
Additionally, the insert was cleaved out of the cloned TA vector by using restriction endonucleases Nde I and Eco RI contained in the aforementioned individual PCR primers, which was then purified; the resulting purified insert was sub-cloned in between the Nde I site and Eco RI site of the pRSET vector (manufactured by Invitrogen Co.) as expression plasmid. The resulting plasmid is a plasmid of the third aspect of the invention.
The plasmid of the third aspect of the invention is used for transformation of expression Escherichia coli , for example Escherichia coli BL21 (DE3) (manufactured by Novagen), to recover a transformant of the fourth aspect of the invention.
Transformation is conducted by general methods including for example calcium chloride method (Cohen, S. N., Chang, A. C. Y. and Hsu, L., Proc. Natl. Acad. Sci. USA, 69: 2110-2114, 1972) and electroporation method (New Experimental Methods For Fundamental Biochemistry, Kinichiro Miura et al. ed., Genetic Engineering, 1988, Maruzen).
The transformed Escherichia coli is deposited as Accession No. FERM BP-6624 at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan).
By culturing the transformant Escherichia coli of the fourth aspect of the invention and identifying the amino acid sequence carried in the Escherichia coli or assaying the specific activity of the enzyme, the expression of the gene of the second aspect of the invention can be confirmed.
Not any plant-derived epoxide hydrolase with a high activity has been demonstrated insofar. The system for expressing such highly active plant-derived epoxide hydrolase at a mass scale is very useful for assessing the function of the enzyme gene in a simple manner.
According to the first and second aspects of the invention, the cDNA encoding the plant-derived epoxide hydrolase and the gene encoding the plant-derived epoxide hydrolase are provided. By using the cDNA and the gene, an epoxide hydrolase gene with significant functions for protecting biological organisms can readily be developed and may be applicable to plants for foods.
According to the third and fourth aspect of the invention, the plasmid vector carrying the cDNA encoding the plant-derived epoxide hydrolase and the transformant retaining the plasmid vector are provided. The transformant can be utilized effectively as an expression system in microorganisms for assessing the functions of the gene encoding the epoxide hydrolase simply and easily.